Cells have ways to direct de novo synthesised proteins to various compartments of the cells and to the extracellular space. Signal peptides are enclosed in the coding part of the chromosomal DNA and are synthesised as part of the protein by the ribosomal apparatus. Signal peptides make up the N-terminal and cause the newly synthesised polypeptides to be directed into the rough endoplasmic reticulum. Here, the signal peptide is cleaved from the polypeptide and the mature protein is secreted into the surroundings. Thus, the signal peptide remains inside the cell.
The pro-part of the protein is cleaved from the mature part of the protein and ends outside the cell. For some neurotrophic factors, e.g. NGF, the pro-part of the protein is bioactive as a neuropeptide.
In gene therapy where the inserted gene codes for a protein which is to be secreted a signal sequence will need to be placed in front of the mature protein to ensure it's proper processing through the rough endoplasmic reticulum and the Golgi Apparatus. The first choice is almost invariably the native signal sequence of the protein in question, because it is generally desired that the protein is secreted and/or processed in the same way as it is secreted and processed by the native cell. For some uses it is also desired that the amount of protein expressed is the same as in the native cell. Furthermore, one cannot exclude the possibility that the cleaved signal sequence plays a role in the metabolism of the cell. Finally one of skill in the art would chose to use the pre-pro-part protein to ensure correct processing and folding of the mature protein.
In many cases it has turned out that in vivo transduced and transfected cells which are supposed to secrete a therapeutic factor do not secrete the therapeutic factor in therapeutically sufficient quantities and for sufficient time. This may also be a problem in ex vivo gene therapy where cells are transfected or transduced outside the body and inserted into the patient after genetic modification.
The prior art does not provide much information concerning the coupling of signal peptide with heterologous proteins in mammals. For heterologous expression of mammalian proteins in fungi or yeast it is common practise to replace the mammalian signal peptide with one that is functional in the producer species.